Blockade of the Epidermal Growth
Factor Receptor Inhibits
Transforming Growth Factor
a-lnduced but Not Estrogen-Induced
Growth of Hormone-Dependent
Human Breast Cancer
Carlos L. Arteaga*, Ester Coronado, and C. Kent Osborne
Department of Medicine
Division of Oncology
University of Texas Health Science Center at San Antonio
San Antonio, Texas 78284-7884
Transforming growth factor a (TGFa), a polypeptide
that binds to the epidermal growth factor (EGF)
receptor, is expressed and secreted by human
breast cancer cells and has been proposed as an
autocrine growth factor and as a mediator of the
mitogenic effect of estrogen. We investigated the
potential importance of secreted TGFa in estrogenresponsive MCF-7 human breast cancer cells using
monoclonal (528ab and 225ab) and polyclonal antibodies that block the EGF/TGFa receptor. Confirming other studies, these MCF-7 cells expressed
TGFa with mRNA transcripts of 4.8 kilobases identified by Northern analysis, and they secreted TGFa
activity measured by normal rat kidney colony-forming assay and an EGF RRA of conditioned medium.
This activity was increased 3-fold by 1 nwi 17/?estradiol and decreased by 1 HM tamoxifen. 528ab
and 225ab bound to EGF receptors in MCF-7 cells
with high affinity [dissociation constant (Kd) 0.1-0.5
nwi] and blocked the binding of EGF/TGFa. These
antibodies failed to inhibit baseline DNA synthesis
or growth of MCF-7 cells although they were potent
inhibitors of EGF/TGFa-induced growth of these
cells. We hypothesized that if secreted TGFa mediates estrogen-induced growth, then EGF/TGFa receptor blockade should inhibit estrogen stimulation.
MCF-7 cells were first treated with tamoxifen to
inhibit growth and to reduce TGFa expression. Under
these conditions, estrogen replenishment induced a
marked dose-dependent rescue of TGFa secretion,
DNA synthesis, and cell proliferation. Exogenous
TGFa also partially restored growth of tamoxifeninhibited cells. Although the simultaneous addition
of 528ab or 225ab blocked TGFa-induced rescue of
MCF-7 cells, it had no effect on rescue by estradiol.
Similar results were observed with a polyclonal antiEGF receptor antibody, and with two other estrogenresponsive breast cancer cell lines. In summary,
blockade of the EGF/TGFa receptor in hormonedependent human breast cancer cells does not alter
estrogen-regulated growth suggesting that secreted
TGFa is not a primary mediator of the growth effects
of estrogen. (Molecular Endocrinology 2:1064-1069,
1988)
INTRODUCTION
Transforming growth factor a (TGFa), a polypeptide
that binds to the EGF receptor (1), has been proposed
as an autocrine growth factor and as a mediator of the
mitogenic effect of estrogen in hormone-dependent
breast cancer cells (2). Several lines of circumstantial
evidence support this hypothesis. First, breast cancer
cells secrete TGFa and secretion is increased by estrogen and decreased by antiestrogens (3, 4). Second,
epidermal growth factor (EGF)-like peptides in media
conditioned by estrogen-primed hormone dependent
breast cancer cells partially support growth of these
cells in ovariectomized nude mice (5). Third, MCF-7
cells transfected with the v-Ha-ras oncogene secrete 3to 4-fold higher levels of TGFa and are tumorigenic in
nude mice in the absence of estrogen supplementation
(6, 7). Fourth, breast cancer biopsies expressing a
larger number of EGF/TGFa receptors tend to lack
estrogen receptors and to behave clinically more aggressively (8), suggesting a role for the EGF/TGFa
receptor (or its ligand) in the progression of breast
cancer. The present studies examine whether secreted
TGFa is an important estrogen-regulated autocrine
growth factor for hormone-responsive human breast
0888-8809/88/1064-1069$02.00/0
Molecular Endocrinology
Copyright © 1988 by The Endocrine Society
1064
TGFa in Hormone-Dependent Human Breast Cancer
1065
that block the EGF/TGFa receptor. We demonstrate
that although blockade of this receptor inhibits TGF«induced growth, it has no effect on baseline cell proliferation or on growth stimulation by estrogen.
I 2 3
-4.8 Kb
RESULTS
TGFa activity, assayed by induction of NRK colonies
and by EGF receptor binding activity, was present in
conditioned medium of MCF-7 cells (Table 1). This
activity was increased 2- to 3-fold in the presence of 1
nM 17j8-estradiol. Tamoxifen, under these estrogen-free
conditions, decreased slightly the level of secreted
TGFa activity measured in the EGF RRA (Table 1).
Consistent with these results, the MCF-7 cells expressed TGFa mRNA transcripts of 4.8 Kb and this
message was increased by estrogen and decreased by
tamoxifen (Fig. 1).
Competitive binding experiments showed that EGF,
TGFa and the receptor antibodies were able to block
binding of 125I-EGF to MCF-7 cells as shown in Fig. 2
for 2 growth factors and the 528ab. Scatchard analysis
of binding data revealed 3500 EGF receptor sites per
cell and a Kd of 0.06, 0.3, 0.5, and 0.1 nM for EGF,
TGFa, 528ab, and 225ab, respectively (data not
shown).
Next we investigated whether 528ab could block the
dose-dependent mitogenic effect of TGFa on MCF-7
cells. TGFa at a concentration of 50 ng/ml (10 nM),
induced a 2-fold increase in the rate of DNA synthesis
in MCF-7 cells growing in phenol red-free serum-free
medium (Fig. 3). Stimulation of DNA synthesis by TGFa
was markedly inhibited by the simultaneous addition of
528ab. An antibody (323A3) raised against a Mr 43,000
membrane glycoprotein in MCF-7 cells (9) was used as
a control. The control antibody did not block the TGFainduced stimulation of DNA synthesis. As shown in Fig.
3, the receptor antibody had no effect on baseline
[3H]thymidine incorporation in the absence of exogenous TGFa. Similar results were observed in monolayer
growth and anchorage-independent growth experi-
Table 1. TGFa Activity in Conditioned Medium of MCF-7
Cells
Control
17/3-Estradiol
Tamoxifen
NRK Colonies/
108 Cells
EGF Equivalents
(pg/106 Cells)
12.3 ± 0 . 3
26.8 ± 4.3fl
12.8 ± 3 . 0
39.1 ± 0.1
98.1 ± 1 . 3 a
29.5 ± 2.6a
Cells were incubated in phenol red-free medium containing
0.1% ethanol (control), 1 nw 17/8-estradiol, or 1 ^ M tamoxifen
for 48 h. Determinations of TGFa activity were done in triplicates in an NRK colony-forming assay and an EGF RRA.
Values were standardized to an equivalent number of viable
cells.
" P < 0.05 compared to control.
-2.0 Kb
Fig. 1. Presence of TGFa mRNA in MCF-7 Cells
A, Polyadenylated RNA was isolated and electrophoresed
in formaldehyde-1.2% agarose gels (5 fig/\ane). Levels of
TGFa mRNA were determined by Northern blot hybridization
from MCF-7 cells grown in media containing 0.1% ethanol
(control) (lane 1), 1 nM estradiol (lane 2), or 1 nM tamoxifen
(lane 3). B, RNA loading was monitored with sequential hybridization with a nick-translated cDNA actin probe which recognized a 2.0 Kb message. The marked decrease in TGFa mRNA
in tamoxifen-inhibited MCF-7 cells (lane 3) could not be accounted for by the difference in RNA load as measured by
densitometry.
lOO-i
80-
Jl«H
Li-
O
w .if
i J 40^
200
10"
I0"' 0
I0" 9
Competitor (M)
10"
10"
Fig. 2. Competition Binding Curves of EGF (•), TGFa (A), and
528ab (O) to MCF-7 Cells
Cells were incubated for 2 h at 22 C with 100 pg/ml 1 2 5 IEGF and different concentrations of unlabeled competitors.
ments (Table 2), suggesting that secreted TGFa does
not contribute significantly to the constitutive rate of
DNA synthesis, cell proliferation, or clonogenicity of the
cells under these in vitro conditions.
Since 528ab inhibited TGFa-induced stimulation of
MCF-7 cells, we hypothesized that antibody blockade
of the EGF/TGFa receptor would also inhibit estrogeninduced stimulation if it is mediated by secreted TGFa
activity. We examined the effect of all three antibodies
on estrogen-induced growth under conditions in which
the effect of estrogen is optimized by first inhibiting the
cells with tamoxifen and then rescuing them with estradiol. Tamoxifen (1 HM) induced a 60% decrease in the
MOL ENDO-1988
1066
Vol 2 No. 11
rate of thymidine incorporation in MCF-7 cells after 48
h (not shown). Exogenous TGFa was capable of partially rescuing the cells from tamoxifen inhibition in a
dose-dependent manner, an effect that was blocked as
expected by 528ab (Fig. 4A). Of note, the degree of
stimulation by TGFa of tamoxifen-inhibited MCF-7 cells
and of noninhibited cells (Fig. 3) was the same. When
the TGFa concentration was kept constant at 50 ng/
ml, the inhibitory effect of 528ab was also dose dependent (Table 3) with an ab concentration of only 2500
ng/ml (17 nM) causing significant inhibition. Estrogen
treatment also rescued the cells from tamoxifen inhibition but, in contrast to that observed with TGFa, estrogen rescue was unaffected by 528ab (Fig. 4B). Similar
experiments were performed using cell number after 5
days in culture as the endpoint for the effect of 528ab
on estrogen-induced growth (Fig. 5). Again, blockade
of the EGF/TGFa receptor inhibited TGFa-induced but
not estrogen-induced cell proliferation.
Experiments using another monoclonal antibody
(225ab) (10) and a polyclonal anti-EGF receptor ab (11)
yielded similar results (Tables 4 and 5). In a second
MCF-7 line (Dr. C. McGrath, Michigan Cancer Foundation, Detroit, Ml), as well as in two other breast cancer
cell lines, T47D, and ZR75-1, the 225ab was also
unable to block estrogen-stimulated DNA synthesis or
cell proliferation (data not shown.).
353025-
Q. 15-
0.5
5.0
50.0
TGFa Concentration (ng/ml)
10"" 1O"10 1O"9 10"8 10"
E2 Concentration (M)
Fig. 4. Rescue of MCF-7 Cells after Antiestrogen-lnduced
Inhibition with TGFa (A) or with Estradiol (E2) (B) in the Presence (O) or Absence (•) of the Anti-EGF/TGFa Receptor
Antibody (15 Mg/ml)
The estrogen rescue experiment was repeated three times
with similar results. The small difference between the curves
at 10" 8 M E2 was not reproduced on any occasion. Rate of
DNA synthesis was measured as described in Materials and
Methods. Data points represent means ± SE of triplicate determinations.
Table 3. Dose-Dependent Inhibition of TGFa Induced
Rescue of MCF-7 Cells Mediated by Anti-EGF/TGFa
Receptor Antibody
TGFa
(ng/ml)
0.5
5.
50
TGFa (ng/ml)
Fig. 3. Effect of TGFa and 528ab on DNA Synthesis in MCF7 Cells
Cells growing in exponential phase in phenol red-free serumfree medium were stimulated with different concentrations of
TGFa (maximum 10 nM) in the presence (O) or absence (•) of
528ab at a concentration of 15 ^g/m\ (100 rriM) or in the
presence of a similar concentration of a control 323A3 ab(A).
Rate of DNA synthesis was estimated at 18 h. Values are the
means ± SE of triplicate determinations.
50
50
50
50
50
50
528ab
(ng/ml)
50
500
2500
5000
15000
[3H]Thymidine
Incorporation
(cpm)
6,448 ± 242
10,244 ±522
9,214 ±327
8,434 ± 472
7,172 ±914
5,574 ± 508
5,059 ±216
After 48 h of tamoxifen-induced inhibition, cells were rescued
with 50 ng/ml TGFa in the presence or absence of various
concentrations of 528ab. After 18 h of rescue, the rate of DNA
synthesis was estimated as described in Materials and Methods. Values represent means of triplicate determinations ± SE.
Table 2. Effect of 528ab on Growth of MCF-7 Cells
Control
528ab(15 M g/ml)
[3H]Thymidine
Incorporation
(cpm)
Monolayer
Growth
(x1CT3
Cells)
Anchorage-Independent
Growth (Colonies)
16,988 ±749
16,465 ±461
401 ± 63
439 ± 1 6
275 ± 29
265 ± 37
[3H]Thymidine incorporation was measured as described in Materials and Methods 18 h after addition of the antibody to exponentially
growing cells. Growth in monolayer was assessed on day 5 after plating 5 x 104 cells in 6-well tissue culture plates with two
exchanges of medium and antibody. Cells were maintained in phenol red-free serum free medium for these two experiments. For
anchorage-independent growth experiments, 3 x 103 cells were plated in 35-mm Petri dishes (Falcon) in 0.8% agarose with phenol
red-free IMEM, 5% CS, 10 mM HEPES. Colonies (>100 n) were counted on day 14. All values represent means of triplicate
determinations ± SE. Each experiment was done twice with similar results.
TGFa in Hormone-Dependent Human Breast Cancer
10"10
10'9
10'8
E2 Concentration (M)
0.5
5.0
50.0
TGFa Concentration (ng/ml)
Fig. 5. Rescue of MCF-7 cells after Antiestrogen-lnduced
Inhibition with TGFa (A) or with Estradiol (E2) (B) in the Presence (O) or Absence (•) of the Anti-EGF/TGFa Receptor
Antibody (15 ^g/ml)
Cell number was assessed after 5 days of rescue. Data
points represent means ± SE of triplicate determinations.
Table 4. Effect of 225ab on Estrogen-Induced Growth of
MCF-7 Cells
[3H]Thymidine
Incorporation
(cpm)
Control
225ab (15 Mg/
E2 (10"9 M)
E2 + 225ab
14,773 ±
16,847 ±
29,155 ±
31,623 ±
Monolayer Growth
(x10~ 3 Cells)
1,808
2,755
546
3,448
110 ±
160 ±
529 ±
432 ±
21
23
85
67a
After 48 h of tamoxifen inhibition, cells were rescued with
estradiol in the presence or absence of 225 ab. The rate of
DNA synthesis and monolayer growth were measured after
18 h and 5 days of rescue, respectively, as described in Figs.
4 and 5. Values represent means of triplicate determinations
±SE.
a
P > 0.10 compared to E2.
Table 5. Effect of Polyclonal Anti-EGF Receptor ab on
Estrogen-Induced Growth of MCF-7 Cells
[3H]Thymidine
Incorporation
(cpm)
Control
ab (1:500)
E2(10"9M)
E2 + ab
TGFa (50 ng/ml)
TGFa + ab
16,013
16,522
31,853
33,130
24,069
16,111
±877
±1,116
±728
±1,334
± 356
± 1,533"
Experiments were identical with those described in Tables 3
and 4. Due to the limited amount of antibody, only the rate of
DNA synthesis was measured. Values represent means of
triplicate determinations ± SE.
a
P < 0.05 compared to TGFa.
DISCUSSION
The MCF-7 cells used in these experiments have EGF/
TGFa receptors, express and secrete TGFa activity,
and are stimulated by exogenous TGFa, suggesting
1067
the possibility that this growth factor might have important autocrine growth activity. To test this hypothesis
we used monoclonal and polyclonal antibodies that bind
to and block the EGF/TGFa receptor thereby inhibiting
the biological effects of these peptides. Although blockade of the EGF/TGFa receptor antagonized growth
stimulation by exogenous TGFa, it did not inhibit basal
DNA synthesis, monolayer growth, or anchorage-independent growth of these cells suggesting that secreted
TGFa activity was not a crucial autocrine growth factor
under these conditions.
Since estrogen treatment caused increased TGFa
expression and secretion, it has been postulated that
estrogen-induced growth is mediated at least in part by
secreted TGFa (2). In order to optimize growth stimulation with estrogen to test this hypothesis in our study
model, we first inhibited MCF-7 cells with the antiestrogen tamoxifen. Antiestrogens induce a transition delay
in early G1 phase that is reversible with estrogen replenishment resulting in a synchronous cohort of cells
progressing into and through S phase (12, 13). The
response of these cells to estrogen without prior antiestrogen inhibition is less consistent and less dramatic,
probably due to the presence of weak estrogenic substances in the culture media in which the cells are
serially passaged (14). Antiestrogen treatment also reduced TGFa secretion (4). Using this model and employing two different growth assays, TGFa was capable
of partially reversing tamoxifen inhibition, an effect
which could be blocked with the EGF/TGFa receptor
antibodies. On the other hand, the receptor antibodies
did not inhibit estrogen-induced rescue of antiestrogeninhibited cells. The mechanism by which TGFa reversed
tamoxifen inhibition is not known. Since TGFa also
controls transit of cells through the cell cycle, it may be
able to override the G, block induced by tamoxifen.
These results suggest that secreted TGFa activity is
not a major mediator of the mitogenic effects of estrogen. One cannot conclude from these data, however,
that secreted TGFa has no effect on estrogen-regulated
growth of these cells. Since other potential growth
factors such as insulin-like growth factor I (15, 16),
insulin-like growth factor II (17), and the cathepsin D
52K protein (18) are also secreted by these cells in
response to estrogen, and since secretion of potential
growth inhibitors such as TGF/3 is decreased by estrogen (19), it is possible that growth regulation by estrogen involves a complex interaction of several secreted
factors. Inhibition of only one of these autocrine loops
may be insufficient to alter estrogen-stimulated growth.
Experiments using combinations of antibodies to neutralize several factors or to block multiple growth factor
receptors will be required to address this possibility.
The recent report that the growth factors secreted
constitutively by MDA-231 cells were not capable of
inducing growth of MCF-7 cells when coinoculated in
castrated female nude mice argues against the hypothesis that these multiple secreted factors can fully mediate estrogen's effects (20). Although secreted growth
factors could partially mediate estrogen-induced
growth, it is possible that estrogen regulation of growth
Vol 2 No. 11
MOL ENDO-1988
1068
is primarily due to other mechanisms such as a direct
effect on key metabolic or DNA-replicating enzymes in
the cell (21), or an indirect effect mediated by the
induction of growth factor production at distant organs
(estromedins) (22), or perhaps by inhibition of a serum
blocking factor (23).
Finally, it is also possible that TGFa activity synthesized by the cell mediates estrogen's effects by interacting with intracellular receptors that would not be
affected by EGF/TGFa receptor antibodies in the culture medium. This possibility requires further study.
Although enhanced TGFa expression and secretion are
clearly associated with estrogen-induced stimulation of
hormone-dependent breast cancer cells (4, 24) as well
as with the acquisition of a more malignant phenotype
(6,7,24), a causal relationship between this polypeptide
growth factor with hormone-regulated growth or with
increased tumorigenicity of breast cancer remains unproved.
in phenol red-free serum-free IMEM containing transferrin (2
mg/liter) and fibronectin (2 mg/liter) (Collaborative Research,
Lexington, MA) for 48 h at 37 C in a 5% CO2 incubator. In
some flasks the IMEM contained 0.1% ethanol, 1 nwi 17/3estradiol (Sigma, St. Louis, MO), or 1 HM tamoxifen (Stuart
Pharmaceuticals, Wilmington, DE). The media were then harvested and Aprotinin (20 TlU/ml) (Sigma) 0.2% vol/vol and
pepstatin, 1 tig/m\ (Boehringer Mannheim Biochemicals, Indianapolis, IN) were added. After centrifugation at 3000 rpm for
15 min, the media were dialyzed against 200 vol 0.1% acetic
acid at 4 C in Spectrapor 3 dialysis tubing (3500 Mr cut-off).
The media were then lyophilized, reconstituted in 10 mM acetic
acid-0.1% BSA, and stored at - 7 0 C until further testing. After
collection of conditioned media, the monolayers were harvested and the cells counted on a hemocytometer for further
standardization of TGFa activity in the media. Viability, as
determined by Trypan blue dye exclusion, was consistently
greater than 90%. TGFa activity in the conditioned media was
determined in anchorage-independent growth assay and a 1 2 5 IEGF RRA as previously described (4, 26). EGF receptor binding activity in the media was calculated by interpolation on the
standard competition curve with unlabeled EGF.
Northern Hybridization
MATERIALS AND METHODS
Cells, Growth Factors, and Antibodies
The MCF-7 cells were kindly supplied by Dr. M. Lippman (NCI)
and T47D cells by Dr. D. Edwards (University of Colorado,
Denver, CO). The ZR75-1 cell line was obtained from the
American Type Culture Collection (Rockville, MD). All breast
cancer cells were passaged in Improved minimum essential
medium (IMEM, Irvine Scientific, Alamosa, CA) supplemented
with 5-10% fetal calf serum (FCS, Gibco Laboratories, Grand
Island, NY), and 10 nM insulin (Eli Lilly Research Laboratories,
Indianapolis, IN). Recombinant human TGFa was provided by
Dr. R. Derynck (Genentech Inc., San Francisco, CA), and EGF
(derived from mouse submaxillary gland) was purchased from
Collaborative Research Laboratories (Lexington, MA). The
528ab and 225ab were generously supplied by Dr. J. Mendelsohn (Memorial Sloan-Kettering Cancer Center, New York,
NY). These monoclonal antibodies inhibit EGF binding in A431
cells and antagonize EGF-stimulated tyrosine protein kinase
activity (10, 25). The polyclonal anti-EGF receptor antibody
(11) was a generous gift from Dr. G. Carpenter (Vanderbilt
University, Nashville, TN). An irrelevant antibody that recognizes another antigenic membrane site in MCF-7 cells (323A3)
was the gift of Dr. W. L. McGuire (University of Texas Health
Science Center at San Antonio) and was used as a control (9).
Receptor Binding Studies
Breast cancer cells were grown to near confluence in 24-well
tissue culture plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ). After two washes with binding buffer (IMEM,
0.1% BSA, 25 rriM HEPES, pH 7.4), the monolayers were
incubated with 100 pg/ml 125I-EGF (Biomedical Technologies,
Inc., Stoughton, MA) plus various concentrations of unlabeled
competitors or different dilutions of conditioned medium. The
incubation was continued for 2 h at 22 C, and then specific
binding was determined as previously described (26).
TGFa Activity of Conditioned Medium
Cells were grown to near confluence in phenol red-free IMEM
supplemented with 5% FCS in T150 flasks. The monolayers
were washed three times with PBS/0.1% BSA and incubated
MCF-7 cells growing in T150 flasks in phenol red-free serumfree IMEM with either 0.1% ethanol, 1 nM 17j8-estradiol, or 1
MM tamoxifen for 48 h were harvested and homogenized and
total RNA was prepared as described (27). Only samples with
nondegraded 28S and 18S ribosomal bands were analyzed.
The polyadenylated RNA fraction was isolated by oligo-dTcellulose chromatography (28). Five micrograms of polyadenylated RNA were electrophoresed into a formaldehyde-1.2%
agarose gel and blotted onto nitrocellulose (29). The nitrocellulose filters were hybridized with a 32P-labeled cDNA TGFa
probe (kindly supplied by the Chiron Corporation, Emeryville,
CA) in 50% formamide, 5x standard saline citrate, 1 x Denhardt's solution, 0.1% sodium dodecyl sulfate, 100 Mg/ml
salmon sperm DNA for 24 h at 50 C. Washings were done in
0.2 x standard saline citrate, 0.2% sodium dodecyl sulfate at
50 C. To monitor RNA load, the nitrocellulose membranes
were sequentially hybridized with a cDNA actin probe which
recognizes a 2.0 Kb message. The relative levels of specific
mRNA were quantified by densitometry.
Cell Proliferation Experiments
MCF-7 cells were plated in 24-well tissue culture plates (Falcon, Oxnard, CA) at a density of 2 x 104 cells per well in IMEM
with 5% FCS. Twenty-four hours later the medium was
changed to phenol red-free IMEM supplemented with 5%
charcoal-stripped calf serum with 1 HM tamoxifen. The purpose
of this step was to reduce baseline TGFa expression and
secretion (4), to inhibit growth, and to block the cells in early
G1 phase in order to magnify the effects of estrogen replenishment (12). Forty-eight hours later, the medium was changed
to phenol red-free IMEM with 5% charcoal-stripped calf serum
with different concentrations of estrogen or of TGFa. Half of
the wells were also treated with 528ab or 225ab at a final
concentration of 15 /*9/ml, or with a 1:500 dilution of the
polyclonal anti-EGF-receptor ab. Eighteen hours later the rate
of DNA synthesis was estimated by measuring [3H]thymidine
incorporation into acid-precipitable material as described previously (15). In other experiments monolayer cell proliferation
was assessed. After tamoxifen inhibition as described above,
the cells were grown in media containing estrogen or TGFa
with or without EGF/TGFa receptor antibody. Fresh medium
with hormones and antibody was exchanged for spent medium
on day 3. On day 5, cells were suspended with 1 mM EDTA
and counted on a hemocytometer.
TGFa in Hormone-Dependent Human Breast Cancer
1069
Statistical Analysis
Statistical evaluation of results was performed with analysis
of variance followed by Neuman-Keul's multiple comparisons.
12.
Acknowledgments
Consultations with Dr. G. M. Clark and the secretarial assistance of Mrs. Pamela Render are greatly appreciated. We thank
Drs. J. Mendelsohn, G. Carpenter, and W. L. McGuire for the
gifts of antibodies.
Received April 5,1988. Accepted July 20,1988.
Address requests for reprints to: Dr. C. Kent Osborne,
Department of Medicine, Division of Oncology, University of
Texas Health Science Center, 7703 Floyd Curl Drive, San
Antonia, Texas 78284-7884.
This work was supported in part by NIH grants R01-CA
30251 (to C.K.O.) and P01-30195 (to C.K.O.) and a VA Career
Development Award (to C.L.A.).
* Recipient of an Associate Investigator Career Development Award from the Veterans Administration.
13.
14.
15.
16.
17.
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